Capillary Electrophoresis

Assuming the separation techniques used in the two dimensions are orthogonal, i.e., the two separation techniques are based on different physicochemic...
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Anal. Chem. 2001, 73, 2669-2674

Two-Dimensional Electrochromatography/Capillary Electrophoresis on a Microchip Norbert Gottschlich, Stephen C. Jacobson, Christopher T. Culbertson, and J. Michael Ramsey*

Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6142

A two-dimensional separation system on a microfabricated device was demonstrated using open-channel electrochromatography as the first dimension and capillary electrophoresis as the second dimension. The first dimension was operated under isocratic conditions, and the effluent from the first dimension was repetitively injected into the second dimension every few seconds. A 25-cm separation channel with spiral geometry for open-channel electrochromatography was chemically modified with octadecylsilane and coupled to a 1.2-cm straight separation channel for capillary electrophoresis. Fluorescently labeled products from tryptic digests of β-casein were analyzed in 13 min with this system. Two-dimensional (2D) separation systems are of interest because of their increased peak capacity over one-dimensional separations. Assuming the separation techniques used in the two dimensions are orthogonal, i.e., the two separation techniques are based on different physicochemical properties of the sample, the peak capacity of the 2D method is the product of the peak capacities of the independent one-dimensional methods,1 and the resolution is the square root of the sum of the squares of the resolution in the two embedded systems.2 2D separation methods for the analysis of complex protein or peptide mixtures are commonly performed on planar gels using isoelectric focusing for the first dimension and polyacrylamide gel electrophoresis for the second dimension (IEF-PAGE).3 However, this technique is slow and labor intensive. Therefore, several column-based twodimensional separation schemes have been developed in order to help reduce the analysis time and labor.4 For these systems, several interfaces have been designed to inject the effluent from the first-dimension column into a second dimension including automated switching valves,5 parallel columns in the second dimension,6 flow gating,7,8 and optical gating.9 Two complementary techniques that can be coupled together relatively easily are (1) Giddings, J. C. Unified Separation Science; John Wiley & Sons: New York, 1991. (2) Giddings, J. C. J. High Resolut. Chromatogr. Chromatogr. Commun. 1987, 10, 319-323. (3) O’Farrell, P. H. J. Biol. Chem. 1975, 250, 4007-4021. (4) Liu, Z.; Lee, M. L. J. Microcolumn Sep. 2000, 12, 241-254 (5) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990, 62, 978-984. (6) Opiteck, G. J.; Jorgenson, J. W.; Anderegg, R. J. Anal. Chem. 1997, 69, 2283. (7) Lemmo, A. V.; Jorgenson, J. W. Anal. Chem. 1993, 65, 1576-1581. (8) Hooker, T. F.; Jorgenson, J. W. Anal. Chem. 1997, 69, 4134-4142. (9) Moore, A. W., Jr.; Jorgenson, J. W. Anal. Chem. 1995, 67, 3448-3455. 10.1021/ac001019n CCC: $20.00 Published on Web 04/26/2001

© 2001 American Chemical Society

microcolumn reversed-phase liquid chromatography and capillary electrophoresis.8 Two-dimensional systems where the separation techniques are coupled in series can be categorized by the number of samples and the fraction of effluent that is sampled by the second dimension. “Heart-cutting” methods select a region of interest from the first dimension and inject that region into the second dimension. In contrast, “comprehensive” two-dimensional systems sample the effluent from the first dimension into the second dimension at regular intervals and fixed volumes. Effluent from the first dimension should be transferred to the second dimension without being diluted or dispersed. The contribution to zone broadening due to finite volume sampling of the first dimension into the second dimension was investigated by Murphy et al.10 To obtain high resolution for comprehensive analysis, each peak from the first dimension should be sampled three times into the second dimension when the starting time for sample collection is in phase with the peak arrival. For sampling that is out of phase with the peak arrival, the effect on the resolution is insignificant when at least four samples are taken across the peak from the first dimension. Microfabricated fluidic devices (microchips) are potentially useful for multidimensional separations because high-efficiency separations can be achieved and small sample volumes can be manipulated with minimal dead volumes between interconnecting channels. Electrokinetically driven separation techniques demonstrated on microchips include capillary electrophoresis (CE),11-14 micellar electrokinetic chromatography (MEKC),15,16 electrochromatography,17,18 and gel electrophoresis.19-22 Recently, a micro(10) Murphy, R. E.; Schure, M. R.; Foley, J. P. Anal. Chem. 1998, 70, 15851594. (11) Harrison, D. J.; Manz, A.; Fan, Z.; Lu ¨ di, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926-1932. (12) Harrison, D. J.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895-897. (13) Effenhauser, C. S.; Manz, A.; Widmer, H. M. Anal. Chem. 1993, 65, 26372642. (14) Jacobson, S. C.; Culbertson, C. T.; Daler, J. E.; Ramsey, J. M. Anal. Chem. 1998, 70, 3476-3480. (15) von Heeren, F.; Verpoorte, E.; Manz, A.; Thormann, W. Anal. Chem. 1996, 68, 2044-2053. (16) Kutter, J. P.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1997, 69, 51655171. (17) Kutter, J. P.; Jacobson, S. C.; Matsubara, N.; Ramsey, J. M. Anal. Chem. 1998, 70, 3291-3297. (18) He, B.; Tait, N.; Regnier, F. Anal. Chem. 1998, 70, 3790-3797. (19) Woolley, A. T.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1134811352. (20) Effenhauser, C. S.; Paulus, A.; Manz, A.; Widmer, H. M. Anal. Chem. 1994, 66, 2949-2953.

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fluidic device for 2D separations of peptide mixtures using MEKC in the first dimension and CE in the second dimension was demonstrated.23 This system connected the two dimensions serially and repetitively injected the effluent of the first dimension into the second dimension by modulating the applied potentials. In contrast, a microfabricated device has been proposed with a single channel for the first dimension and for the second dimension an array of 500 parallel channels positioned orthogonally to the first dimension channel.24 Electrokinetically driven separation techniques eliminate the need for pumps and, therefore, enable a simple instrument design. In addition, the flat flow profile in an electrokinetically driven system reduces band broadening compared to a parabolic (Poiseuille) flow profile in pressure-driven systems.25 In a chromatographic system, if the operating parameters remain unchanged, the number of theoretical plates increases linearly with increasing separation length. To maintain a compact microchip geometry and increase the separation channel length, turns need to be introduced into the channel but can contribute to the band broadening. This geometric dispersion or “racetrack” effect is related to the square of the channel width and the inverse of the radius of curvature of the turn.26 When the channel width cannot be easily reduced, a spiral channel design with a large radius of curvature helps minimize the geometric dispersion.27 An alternative approach to control the geometric dispersion is to taper the channel width in a turn. Low dispersion turns have been reported for symmetrically28 and asymmetrically29,30 tapered turns. In this paper, open-channel electrochromatography (OCEC) and capillary electrophoresis were serially coupled to analyze a tryptic digest of β-casein. A microchip with a 25-cm spiral separation channel for the first dimension was connected to a 1.2cm straight separation channel for the second dimension (Figure 1). For sampling of the effluent from the OCEC channel, small aliquots were repetitively transferred into the CE channel at regular intervals. The volume injected into the second dimension was ∼9% of the total elution volume of the first dimension. EXPERIMENTAL SECTION Chemicals. The separation buffer in all experiments was 10 mM sodium tetraborate (EM Science, Gibbstown, NY) and 30% (v/v) acetonitrile (J. T. Baker, Phillipsburg, NJ) with 0.1% (v/v) trifluoroacetic acid (TFA; Applied Biosystems, Foster City, CA). β-Casein, bovine trypsin (EC 3.4.21.4), and L-phenylalanine were obtained from Sigma (St. Louis, MO). The labeling reagent tetramethylrhodamine isothiocyanate (TRITC) isomer-5 was purchased from Molecular Probes, Inc. (Eugene, OR), and a stock (21) Woolley, A. T.; Mathies, R. A. Anal. Chem. 1995, 67, 3676-3680. (22) Waters, L. C.; Jacobson, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R. S.; Ramsey, J. M.. Anal. Chem. 1998, 70, 158-162. (23) Rocklin, R. D.; Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 2000, 72, 52445249. (24) Becker, H.; Lowack, K.; Manz, A. J. Micromech. Microeng. 1998, 8, 24-28. (25) Martin, M.; Guiochon, G. Anal. Chem. 1984, 56, 614-620. (26) Culbertson, C. T.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1998, 70, 3781-3789. (27) Culbertson, C. T.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 2000, 72, 5814-5819. (28) Paegel, B. M.; Hutt, L. D.; Simpson, P. C.; Mathies, R. A. Anal. Chem. 2000, 72, 3030-3037. (29) Griffiths, S. K.; Nilson, R. H. Anal. Chem. 2000, 72, 5473-5482. (30) Molho, J. I.; Herr, A. E.; Mosier, B. P.; Santiago, J. G.; Kenny, T. W.; Brennen, R. A.; Gordon, G. B.; Mohammadi, B. Anal. Chem. 2001, 73, 1350-1360.

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Figure 1. Image of the microchip used for 2D separations. The separation channel for the first dimension (OCEC) extends from the first valve V1 to the second valve V2. The second dimension (CE) extends from the second valve V2 to the detection point y. Sample (S), buffer 1 and 2 (B1, B2), sample waste 1 and 2 (SW1, SW2), and waste (W) reservoirs are positioned at the terminals of each channel. The arrows indicate the detection points in the OCEC channel (x) and CE channel (y). The channels and reservoirs are filled with black ink for contrast.

solution at 10 mg/mL was prepared in dimethyl sulfoxide (DMSO) (Fisher Scientific, Fair Lawn, NJ). The silanizing reagent octadecyltrimethoxysilane was purchased from Gelest (Tullytown, PA), and rhodamine B was purchased from Eastman Kodak (Rochester, NY). Sample Preparation. β-Casein was dissolved in 100 mM sodium tetraborate at 10 mg/mL, digested with trypsin for 18 h at room temperature (the weight ratio of substrate to enzyme was ∼50), and then filtered (0.2 µm). For the fluorescent labeling of the β-casein digest, 25 µL of TRITC stock solution was combined with 75 µL of the protein digest. The labeling reaction was allowed to proceed for 4 h at room temperature in the dark. TRITC was in molar excess to the undigested protein by a factor of 18. The labeled peptide mixtures were diluted 10-fold for the separation experiments. TRITC-labeled phenylalanine was prepared by dissolving the amino acid in 100 mM sodium tetraborate at 25 mM. An 80-µL aliquot of this solution was combined with 20 µL of the TRITC stock solution and reacted for 4 h at room temperature. Microchip Device. The microchip used in this study was fabricated from white crown glass (Hoya Corp. USA, Shelton, CT) using standard photolithography, wet chemical etching, and bonding procedures.31 The photomask for the microchip in Figure 1 was designed in-house using MiniCAD (Diehl Graphsoft, Inc; Columbia, MD) and fabricated by HTA Photomask (San Jose, CA). The channel design was transferred onto the glass substrates using positive photoresist and UV flood exposure (360-440 nm for 30 s). After developing the photoresist (MF319, Shipley), the chrome film was etched (CeSO4/HNO3; Transene Co.), and then the channels were etched into the substrate in a buffered oxide etch (HF/NH4F, Transene Co.). Channel access holes (2 mm in diameter) were ultrasonically drilled through the substrates at the terminals of the channels. To form the closed network of channels, (31) Jacobson, S. C.; Hergenro¨der, R.; Koutny, L. B.; Warmack, R. J.; Ramsey, J. M. Anal. Chem. 1994, 66, 1107-1113.

the cover plate was bonded to the substrate over the etched channels by hydrolyzing both surfaces (NH4OH/H2O2; J. T. Baker/EM Science), rinsing with water, bringing the cover plate into contact with the substrate, and annealing to 500 °C. Sample, buffer, and waste reservoirs were glued to the chip using epoxy (353ND-T; Epoxy Technology, Billerica, MA). Figure 1 shows a scanned image of the assembled microchip with the channels and reservoirs filled with black ink for contrast. The channel lengths were 25 cm for the OCEC channel and 1.2 cm for the CE channel. The narrow channels were 10.8 µm deep and 34 µm wide at halfdepth, and the wide channels were 10.8 µm deep and 212 µm wide at half-depth. For coating of the OCEC channel,13 the microchip was prepared by sequentially rinsing with 1 M sodium hydroxide solution, water, 1 M hydrochloric acid, water, and methanol (J. T. Baker) and then dried at 110 °C overnight. Toluene (EM Science) was dried over 3-Å molecular sieve (EM Science). Both a 10% (w/w) solution of octadecyltrimethoxysilane and a 1% (v/ v) solution of n-butylamine (Sigma) were prepared with dried toluene. The coating solution was then prepared by adding 10 µL of the n-butylamine solution to 1 g of the octadecyltrimethoxysilane solution. To coat the spiral channel of the microchip with this C18 phase and leave the CE channel uncoated, vacuum was applied at the buffer 2 reservoir, and the coating solution was added to the sample, buffer 1, and sample waste 1 reservoirs. The sample waste 2 and waste reservoirs were filled with neat toluene. After 1 h at room temperature, the coating solution was washed out of the channels with toluene. The microchip was then rinsed with methanol and dried. Microchip Operation. High voltage was applied to the reservoirs of the microchip with five independently controlled high-voltage power supplies (10A12-P4, UltraVolt, Ronkonkoma, NY). Two high-voltage relays (K81C245, Kilovac, Santa Barbara, CA) were connected in series between the high-voltage supplies and the buffer 2 and sample waste reservoirs to remove the high voltage at these reservoirs. Electrical contact between the solutions in the fluid reservoirs and the high-voltage leads was achieved using platinum wires. The high-voltage supplies and relays were computer controlled using a multifunction I/O card (PCI-MIO16XE50, National Instruments, Austin, TX) and LabView (National Instruments). For the first dimension of the 2D separations, a single gated injection32 of sample was made into the OCEC channel at valve V1 to start the run, and for the second dimension, a series of gated injections was made into the CE channel at valve V2 to rapidly sample the effluent from the OCEC channel. Figure 2 shows a timing diagram for the two-dimensional separation with a 0.5-s injection into the OCEC channel, 0.2-s injections into the CE channel, and a 3.0-s delay between injections for the CE channel. In the run mode, the potentials applied to the sample, buffer 1, sample waste 1, buffer 2, sample waste 2, and waste reservoirs were 9.5, 10.0, 5.0, 3.0, 2.5, and 0.0 kV, respectively. With these applied potentials, the field strengths were 220 V/cm in the OCEC channel and 1890 V/cm in the CE channel. In the run mode, the sample was transported down the sample channel through valve V1 into the sample waste 1 channel. The potential applied to the (32) Jacobson, S. C.; Koutny, L. B.; Hergenro¨der, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66, 3472-3476.

Figure 2. Timing diagram for making the initial 0.5-s injection into the OCEC channel (solid line) and the subsequent 0.2-s injections into the CE channel with a cycle time of 3.2 s (dashed line).

buffer reservoir prevented sample transport into the OCEC channel. To initiate the start of a separation sequence, sample was electrokinetically injected into the OCEC channel by lowering the potential applied to the buffer 1 reservoir from 10.0 to 8 kV for 0.5 s. To terminate transport of sample into the OCEC channel, the potential at the buffer reservoir was returned to 10.0 kV. In the inject mode, the field strength in the OCEC channel was 190 V/cm. After the initial injection of sample into the OCEC channel, a series of gated injections of the effluent from the OCEC channel were made into the CE channel at a fixed interval, i.e., every 3.2 s. The gated injections at valve V2 into the CE channel were performed slightly differently using high-voltage relays to enable faster gating of the OCEC effluent into the CE channel. The potentials at the buffer 2 and sample waste 2 reservoirs were applied through two high-voltage relays. To make an injection, the relays were opened for 0.2 s to remove the potentials applied to the buffer 2 and sample waste reservoirs. With the relays open, the OCEC effluent was transported into the CE channel. At the end of the 0.2-s injection period, the relays were closed to terminate transport of sample into the CE channel. During injections at valve V2, the field strengths in the OCEC and CE channels were equal at 320 V/cm. Laser-induced fluorescence (LIF) detection was conducted in either the OCEC channel (detection point x in Figure 1) or the CE channel (detection point y in Figure 1). The beam of an argon laser (514 nm, 543-AP-A01, Omnichrome, Chino, CA) was focused onto the microchip using a 200-mm-focal length lens. The fluorescence signal was collected with a 40× microscope objective and registered by a photomultiplier tube (77348, Oriel, Stratfort, CT) after being spatially (0.6-mm-diameter pinhole) and spectrally (580DF30 band-pass, Omega Optical, Battleboro, VT) filtered. The signal from the photomultiplier tube was amplified (SR570, Stanford Research systems, Sunnyvale, CA) and digitized using the same multifunction I/O card and LabView program. Data were collected continuously from the start of the OCEC separation, and two-dimensional plots of the data were generated by dividing the temporal signal into successive runs for each CE injection and plotting each electropherogram at the corresponding time on the OCEC axis. This procedure was written using IGOR Pro (WaveMetrics, Lake Oswego, OR). RESULTS AND DISCUSSION First, the sampling of the effluent from the OCEC channel into the CE channel was tested. Rhodamine B (10 µM) was continuously fed into the OCEC channel, and after the sample reached Analytical Chemistry, Vol. 73, No. 11, June 1, 2001

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Figure 3. Contour plot of 200 injections into the CE channel. The sample, rhodamine B (10 µM) in 10 mM sodium borate with 30% (v/v) acetonitrile, was continuously infused through the OCEC channel and injected into the CE channel at valve V2.

Figure 5. (a) Gaussian concentration profile (G) and the reconstructed concentration profiles resulting from 1, 2, and 6 samples per peak. (b) Variation of the standard deviation of the sample peak relative to a Gaussian peak with the number of samples per peak.

Figure 4. Variation of the OCEC plate height with injected sample concentration of TRITC-labeled phenylalanine. The buffer was 10 mM sodium borate with 30% (v/v) acetonitrile.

valve V2, 200 injections were repetitively made into the CE channel with a period of 10 s and an injection time of 0.5 s and detected at point y (Figure 1). By stacking each successive CE run at the appropriate time on the first-dimension axis, a 2D contour plot was generated as shown in Figure 3. The relative standard deviation (RSD) in migration time was 1.9% due to a gradual increase in the electroosmotic flow in the CE channel rather than a random variation. The RSD in peak area was 5.5% for this experiment. To test the amount of sample that could be loaded onto the OCEC channel without a significant loss of efficiency, different concentrations of TRITC-labeled phenylalanine were injected into the OCEC channel, and the efficiency was measured. The detection point was positioned at the end of the OCEC channel at detection point x (see Figure 1). Figure 4 shows the variation of the OCEC separation efficiency with injected sample concentration. For concentrations of the phenylalanine of 11 µM and below, an average plate height of 1.4 µm was calculated. A rapid loss in efficiency was observed for TRITC-labeled phenylalanine concentrations greater than 100 µM. Because fluorescence detection was 2672 Analytical Chemistry, Vol. 73, No. 11, June 1, 2001

used, high sample concentrations were not necessary, and the microchip was operated with sample concentrations in a range where good efficiencies could be achieved. The plate heights obtained in the OCEC channel compare well with 2-µm plate heights previously reported for 5.2-µm-deep, straight channels on microchips13 and also for CEC performed in capillaries.33 Another important aspect when separation techniques are serially coupled is making a representative injection from the first dimension into the second dimension. For each profile eluting from the first dimension, the sampling rate or number of samples per peak must be at least one to transfer the sample to the second dimension. We considered a Gaussian concentration profile eluting from the first dimension with a baseline width of 8σ, and the injection time for each sample into the second dimension was short compared to the temporal duration of the Gaussian peak. The first-dimension peak can be reconstructed by interpolating the concentration between each sampling point. The Gaussian concentration distribution along with the reconstructed peaks is shown in Figure 5a for 1, 2, and 6 samples per peak. For this exercise, sampling started exactly when the peak arrived (-4σ), and samples taken at -4σ and 4σ were not counted in number of samples taken across the peak. The standard deviation of the (33) Tan, Z. J.; Remcho, V. T. Anal. Chem. 1997, 69, 581-586.

Figure 6. 2D separation of TRITC-labeled tryptic peptides of β-casein. The projections of the 2D separation into the first dimension (OCEC) and second dimension (CE) are shown to the left and below the 2D contour plot, respectively. The field strengths were 220 V/cm in the OCEC channel and 1890 V/cm in the CE channel. The buffer was 10 mM sodium borate with 30% (v/v) acetonitrile. The detection point y in Figure 1 was 0.8 cm past valve V2 in the CE channel.

reconstructed peak (σs) was calculated by taking the square root of its second moment. The standard deviation of the reconstructed peak relative to the standard deviation of the original Gaussian concentration distribution (σs/σ) increased with decreasing sample number (Figure 5b). For sampling rates of 3, the profile was adequately sampled although the reconstructed profile appeared distorted (Figure 5a). Sampling in phase may not be likely during an experimental run. However, the effect of the sampling phase was insignificant when four or more samples were taken across the peak. These results corresponded with the findings of Murphy et al.,10 where the effluent from the first dimension was stored in a sample loop and then delivered to the second dimension. Figure 6 shows a two-dimensional contour plot of the separation of TRITC-labeled peptides from a tryptic digest of β-casein. Also shown are a reconstructed chromatogram and electropherogram obtained by projecting the data from the contour plot into their respective axes. For this experiment, an injection of the effluent of the OCEC channel into the CE channel was made every 3.2 s, and the sample was detected 0.8 cm into the CE channel at detection point y. In Figure 6, only the data from 300 to 800 s in the OCEC separation and from 1.0 to 2.0 s in the CE separation are shown because 95% of the information obtained fell within this time window. Across the 300-800-s elution window, the peaks eluting from the OCEC channel had an average baseline width of 6 s, and on average, two samples per peak were injected into the CE channel. With the experimental apparatus used, a higher sampling rate was not possible. Approximately 9% of the effluent from the OCEC channel was sampled by the CE channel. This was estimated by taking the ratio of the field strengths in the

Figure 7. One-dimensional OCEC separation of TRITC-labeled tryptic peptides of β-casein. The field strength was 220 V/cm in the OCEC channel. The buffer was 10 mM sodium borate with 30% (v/ v) acetonitrile. The detection point x in Figure 1 was 24 cm past valve V1 in the OCEC channel.

OCEC channel during injection into the CE channel (330 V/cm) and run mode in the OCEC channel (220 V/cm), multiplying by the injection time into the CE channel (0.2 s), and dividing by the cycle time of the CE run (3.2 s). Defining a signal threshold of 10% of the highest detected peak, 26 spots could be distinguished in the 2D plot for the separation of the β-casein tryptic digest. Using the same 10% threshold, 17 peaks were counted in an OCEC run (Figure 7) under the same elution conditions but detected at point x. A total of 50% more peaks were observed in the 2D separation compared to the OCEC separation. If the tryptic digest of β-casein was complete, the maximum number of resolved components expected would be 16 peptides. The increase from the expected 16 peaks to the observed 26 peaks in the 2D separation was probably due to incomplete digestion of the protein and multiply labeled products. Although the digestion and labeling were not optimized, the sample did provide a sufficiently complex mixture with which to test the 2D system. In Figure 6, some of the spots in the 2D plane laid on a diagonal indicating a correlation between the separation mechanisms of the two dimensions. Ideally, the individual separation techniques used in a 2D system should be orthogonal to reduce redundancy in the system and maximize the amount of information obtained. OCEC is a hybrid of reversed-phase liquid chromatography and capillary electrophoresis where the analytes are transported through the channels electrokinetically and separated by hydrophobic and electrophoretic means. The second-dimension CE relies solely on differences in electrophoretic mobility. Using the data in Figure 6, a peak capacity of 150 can be estimated for this 2D system using a peak capacity of 30 for the first-dimension OCEC and 5 for the second-dimension CE. Due to the correlation between OCEC and CE, the entire 2D surface was not accessible, and consequently, the peak capacity was lower than can be achieved in a system with truly orthogonal separation methods. In conclusion, the information content increased by ∼50% going from the one-dimensional OCEC separation (Figure 7) to the twoAnalytical Chemistry, Vol. 73, No. 11, June 1, 2001

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dimensional OCEC/CE separation (Figure 6). Higher sampling rates of the effluent of the first dimension into the second dimension can further improve overall resolving power. Also, photopolymerization may allow the fabrication of 2D microchip structures with packed beds or monolithic stationary phases34,35 to increase the separation performance and sample loading of the chromatographic dimension. Although the two separation strategies integrated on this microchip were not fully orthogonal, this approach might be useful for rapid, automated fingerprinting of proteins and protein digests with possible coupling to mass spectrometric detection. (34) Ericson, C.; Holm, J.; Ericson, T.; Hjerte´n, S. Anal. Chem. 2000, 72, 81-87. (35) Chen, J.-R.; Dulay, M. T.; Zare, N.; Svec, F.; Peters, E. Anal. Chem. 2000, 72, 1224-1227.

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ACKNOWLEDGMENT Research sponsored by National Cancer Institute under Grant R33CA83238. Oak Ridge National Laboratory is managed and operated by UTsBattelle, LLC under Contract DE-AC05-00OR22725 with the U.S. Department of Energy. This research was supported in part by an appointment for N.G. to the ORNL Postdoctoral Research Associates Program, administered by ORISE and ORNL. The authors thank Christopher D. Thomas for preparation of the microchips. Received for review August 24, 2000. Accepted March 20, 2001. AC001019N